Fragmentation of proteins in proteomic research
by Harald Zähringer, Labtimes 05/2010
Passing vegetables through a food mill.
Cooks use food mills to separate the pulp and juice of fruits from skin and seeds or to cream soups. The same principle, however, also works in proteomic research to fragment proteins.
Proteomic researchers usually rely on liquid chromatography-mass spectrometry (LC-MS) to identify and quantify the myriads of proteins that make up the proteome of a single cell or a certain tissue. Usually, the extracted proteins are enzymatically digested and the resulting peptide fragments are subsequently injected into the LC-MS system. The two most popular methods for protein fragmentation are In-gel and In-solution enzymatic digestion; both methods, however, have their drawbacks. Membrane proteins, especially, may get lost during the preparation process, since membrane-spanning segments may not be accessible to the proteolytic enzymes.
Jacek Wiesniewski and his colleagues from Matthias Mann’s group at the Max Planck Institute of Biochemistry in Martinsried, Germany thus thought of and developed an alternative protein fragmentation method called Filter Aided Sample Preparation (FASP). The principle of FASP is akin to a food mill that gets used in the kitchen to pass soups or fruits through a fine-meshed sieve. Instead of a food mill, however, Wiesniewski et al. use an ultrafiltration device and pass the protein fragments through the nanopores of a microcon membrane (you may download detailed FASP instructions from the Mann group Website (www.biochem.mpg.de/mann/approaches/fasp/index.html).
Though the protein extraction method that precedes the FASP protocol may slightly differ from lab to lab, depending on the cells or tissues under study, the extraction protocol usually calls for heating the cells or tissues in the presence of high SDS concentrations and reducing disulphide bridges with dithiothreitrol (DTT). To get rid of the SDS binding to the proteins, the extract is loaded onto the ultrafiltration filter device and SDS is washed away by repeated centrifugation at 14,000 x g in the presence of 8M urea. While small molecules, such as SDS and urea pass the filter, proteins are retained on the filter membrane and may be enzymatically digested in the next step. The digestion is performed directly on the filter unit by adding a trypsin solution and incubation for 4 to 18 hours at 37 °C. The filter unit is installed into a new tube and the attached peptides are forced through the membrane by centrifugation at 14,000 x g for 10 minutes. The highly pure peptides collected in the centrifuge tube are easily separated on HPLC columns and may subsequently be injected into the mass spectrometer.
In a very recent paper, the Mann group describes a FASP variant for processing formalin-fixed and paraffin-embedded (FFPE) tissue samples, prior to mass spectroscopic proteome analysis (Ostasiewicz et al. J. Proteome Res. 2010 Jul 2;9(7):3688-3700). FFPE biobanks have gained some interest in proteome research as an alternative to fresh or frozen tissue but analysis of posttranslational modifications of proteins fixed in FFPE samples was thought to be impossible. Ostasiewicz and his colleagues, however, showed that post-translational modifications such as phosphorylation and N-glycosylation are preserved if the proteins are extracted according to the FASP method.
The FASP-FFPE extraction starts with repeated incubations of FFPE slices in xylene and absolute ethanol. The samples are, subsequently, vacuum-dried, mixed with lysis buffer (0.1 M Tris-HCl, pH 8.0, 0.1 M DTT) and homogenised on ice. SDS is added, after a brief sonication step to a final concentration of 4% and the suspension is heated at 99°C for one hour. After cooling down to room temperature, the protein extracts are centrifuged at 16,000 x g for 10 minutes. The subsequent steps to remove SDS and to digest the proteins are basically the same as for the standard FASP protocol described above.
Last Changed: 10.11.2012